Therapeutic drug for lipid-peroxidation-induced diseases and screening method for therapeutic drugs for lipid-peroxidation-induced diseases

ABSTRACT

The present invention provides: an assay method that uses a compound represented by formula (I) as a fluorescent probe molecule and that is for detecting the lipid peroxidation suppression activity of a test compound; an assay kit that uses the assay method; a screening method that uses the assay method; and a pharmaceutical composition that is for the treatment, etc. of diseases (such as age-related macular degeneration) that are induced by lipid peroxidation reactions.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. national stage of application No. PCT/JP2018/025496,filed on Jul. 5, 2018. Priority under 35 U.S.C. § 119(a) and 35 U.S.C. §365(b) is claimed from Japanese Patent Application No. 2017-132772,filed Jul. 6, 2017; the disclosures of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention provides assay methods and assay kits forexploring lipid peroxidation inhibitors, screening methods for lipidperoxidation inhibitors, and therapeutic drugs for lipid peroxidationreaction-induced diseases.

BACKGROUND ART

Diseases involving lipid radicals in lipid peroxidation reactions span awide range of disease areas such as the cardiovascular system, thecentral nervous system, the respiratory system, and antibacterial drugs(Non Patent Literature 1) (see FIG. 1 ). However, for the diseasescaused by lipid peroxidation reaction, a large number of lipid peroxidesand their metabolites are involved in each disease. Thus, it is not easyto explore useful therapeutic drugs for each disease. Until now, manyantioxidants have been known, but few have been approved aspharmaceuticals. Thus, it is required to provide a drug that exhibitslipid peroxidation reactions inhibitory effects.

As methods for exploring an active drug, screening methods are known.Several methods (for example, TBARs method) are known as methods formeasuring lipid peroxidation reaction inhibition. However, these methodshave problems such as a wide range of objects to be measured; theinability to measure samples of different absorption wavelengths whenutilizing the principles of fluorescence or absorption; or thecomplexity of procedures such as pH manipulation or heating. Thus, thereis a demand for the establishment of an assay method that specializes inthe detection of lipid peroxidation reactions and that allowsmulti-analyte analysis under mild conditions close to those of livingorganisms.

The inventors of the present invention have so far developed excellentprofluorescent nitroxide probe compounds capable of capturing lipidradicals (Patent Literature 1).

Age-related macular degeneration (AMD) is known to be a disease withhigh unmet medical needs in which treatment satisfaction is low andcontribution of drugs for the treatment is low. Age-related maculardegeneration is categorized based on pathogenic mechanism into twotypes, the atrophic (dry) and the exudative (wet). In the United States,atrophic (dry) patients accounts for a large proportion of about 85% toabout 90%, while in Japan, exudative (wet) patients accounts for a largeproportion of about 92%. However, effective therapeutic drugs for theatrophic (dry) disease are not known. Moreover, therapeutic drugseffective for treating and suppressing progression of age-relatedmacular degeneration have not been developed from antioxidants.

CITATION LIST Patent Literature

-   Patent Literature 1: Japanese Patent Application No. 2017-090739

Non Patent Literatures

-   Non Patent Literature 1: Frijhoff J et al., Antioxid. Redox Signal,    2015, 23 (14), 1144-70

SUMMARY OF INVENTION Technical Problem

The present invention provides assay methods and assay kits fordetecting lipid peroxidation inhibition, using profluorescent nitroxideprobe compounds. The present invention further provides screeningmethods using these assay methods. The present invention also providespharmaceutical compositions for treating lipid peroxidationreaction-induced diseases, such as age-related macular degeneration,using the active drug found by the screening methods of the presentinvention.

Solution to Problem

The present inventors have intensively investigated assay methods andscreening methods for detecting and evaluating lipid peroxidationinhibition, and as a result, have found that assay methods usingprofluorescent nitroxide probe compounds and screening methods using theassay methods can readily explore candidate compounds that exhibit lipidperoxidation inhibitory activity. The present inventors have also foundthat these candidate compounds are useful for the treatment orprevention of diseases caused by lipid peroxidation reactions,particularly age-related macular degeneration.

That is, the present invention provides the following aspects, but isnot limited thereto.

(Assay Kits and Assay Methods)

Item [1] An assay kit for detecting lipid peroxidation inhibitoryactivity of a test compound, comprising:

a compound represented by formula (I):

a liposome, andat least one compound selected from the group consisting of2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ionsource materialin a buffer.

Item [1-2] The assay kit according to item [1], wherein the compoundrepresented by the formula (I) has a concentration of 1.0 to 20.0 μM,

the liposome is prepared from egg yolk-derived phosphatidylcholine anddihexadecyl hydrogen phosphate, the egg yolk-derived phosphatidylcholinehas a concentration of 5.0 to 10.0 mg/mL, the dihexadecyl hydrogenphosphate has a concentration of 0.01 to 1.0 mg/mL, the test compoundhas a concentration of 5 to 100 μM,the 2,2′-azobis(2-aminopropane) dihydrochloride has a concentration of 5to 50 mM, and the divalent iron ion source has a concentration of 0.5 to5 mM.

Item [2] The assay kit according to item [1], wherein the divalent ironion source material is iron(II) sulfate.

Item [3] The assay kit according to [1] or [2], comprising a packageinsert showing an activity value of a compound having lipid peroxidationinhibitory activity.

Item [4] An assay kit for detecting lipid peroxidation inhibitoryactivity of a test compound, comprising:

a compound represented by formula (I):

a cultured cell, andat least one compound selected from the group consisting of arachidonicacid and tert-butyl hydroperoxidein a buffer.

Item [4-2] The assay kit according to item [4], wherein the compoundrepresented by the formula (I) has a concentration of 1.0 to 20.0 μM;

the cultured cell has a concentration of 1×10⁴ to 1×10⁵ cells,

the test compound has a concentration of 5 to 500 μM,

the arachidonic acid has a concentration of 100 to 400 μM, and

the tert-butyl hydroperoxide has a concentration of 100 to 400 μM.

Item [5] The assay kit according to item [4], wherein the cultured cellis a human hepatoma-derived HepG2 cell.

Item [6] The assay kit according to item [4] or [5], comprising apackage insert showing an activity value of a compound having lipidperoxidation inhibitory activity.

Item [7] An assay kit comprising any two or more of assay kits accordingto items [1] to [6].

Item [7-2] The assay kit according to item [7], comprising a combinationof:

an assay kit wherein a reaction initiator is 2,2′-azobis(2-aminopropane)dihydrochloride; and

an assay kit wherein a reaction initiator is iron(II) sulfate.

Item [7-3] The assay kit according to item [7], comprising a combinationof:

an assay kit wherein a reaction initiator is arachidonic acid, and

an assay kit wherein a reaction initiator is tert-butyl hydroperoxide.

Item [8] An assay method for measuring lipid peroxidation inhibitoryactivity, comprising:

i) preparing a buffer containing a compound represented by formula (I)and a liposome;

ii) adding at least one compound selected from the group consisting of2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ionsource material;

iii) adding a test compound;

iv) measuring fluorescence; and

v) determining an activity value of the test compound from the result ofmeasuring the fluorescence.

Item [9] An assay method for measuring lipid peroxidation inhibitoryactivity, comprising:

i) preparing a buffer containing a compound represented by formula (I)and a cultured cell;

ii) adding at least one compound selected from the group consisting ofarachidonic acid and tert-butyl hydroperoxide,

iii) adding a test compound;

iv) measuring fluorescence; and

v) determining an activity value of the test compound from the result ofmeasuring the fluorescence.

Item [10] The assay method according to item [8] or [9], comprising

vi) comparing with an activity value of a compound serving as anindicator of lipid peroxidation inhibitory activity.

Item [11] The assay method according to any one of items [8] to [10],for use with a microwell plate.

Item [11-2] The assay method according to item [11], comprising:

i) dispensing a solution of a test compound into the microwell plate;

ii) dispensing a solution containing a compound represented by formula(I) and a liposome or a cultured cell into each well;

iii) when using the liposome, dispensing a solution containing at leastone compound selected from the group consisting of2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ionsource material into the each well, and

when using the cultured cell, dispensing a solution containing at leastone compound selected from the group consisting of arachidonic acid andtert-butyl hydroperoxide into the each well; and

iv) measuring fluorescence with a microplate reader.

(Screening Method)

Item [12] A screening method for selecting a candidate compound havinghigh lipid peroxidation inhibitory activity, comprising:

i) selecting a test compound from a compound library;

ii) performing a screening using the test compound by the assay methodaccording to item [8] using 2,2′-azobis(2-aminopropane) dihydrochloride,and selecting a compound having a high activity value; and

iii) then, performing a screening using the compound having a highactivity value in ii) by the assay method according to item [8] using adivalent iron ion source material, and selecting a compound having ahigh activity value.

Item [13] The screening method according to item [12], when the compoundlibrary is a library containing an unapproved compound as a food orpharmaceutical, further comprising, in addition to the screening methodaccording to item [12]:

i) performing a screening by the assay method according to item [9]using arachidonic acid, and selecting a compound having a high activityvalue;

ii) performing a screening by the assay method according to item [9]using tert-butyl hydroperoxide, and selecting a compound having a highactivity value; and

iii) selecting a compound having high activity values in both of thescreenings of i) and ii).

Item [14] The screening method according to item [12] or [13], when thecompound library is a library containing an unapproved compound as afood or pharmaceutical, further comprising, in addition to the screeningmethod according to item [12]:

i) performing a screening by an assay method according to an MTT methodusing a culture medium containing a cultured cell, a test compound andarachidonic acid, and selecting a compound having high cell viability;

ii) performing a screening by an assay method according to an MTT methodusing a culture medium containing a cultured cell, a test compound, andtert-butyl hydroperoxide, and selecting a compound having high cellviability; and

iii) selecting a compound having high cell viability in both of thescreenings of i) and ii).

Item [15] The screening method according to item [13] or [14],comprising:

i) selecting a structural analog of a compound selected by the screeningmethod according to item [13] or [14] from a compound library;

ii) performing the screening method according to item [13] for thecompound selected in i) and optionally the compound selected by thescreening method according to item [13] or [14], and selecting acompound having a high activity value;

iii) performing the screening method according to item [14] for thecompound selected in i) and optionally the compound selected by thescreening method according to item [13] or [14], and selecting acompound having high cell viability;

iv) selecting a candidate compound having a high activity value and highcell viability in both of the screening methods of ii) and iii);

v) performing an assay method using a culture medium containing acultured cell for the compound selected in i) and optionally thecompound selected by the screening method according to item [13] or[14], and selecting a candidate compound having high cell viability, andvi) selecting a candidate compound from the compound selected in iv) andthe compound selected in v).

Item [16] The screening method according to any one of items [12] to[15], wherein the compound library is Core Library of Drug DiscoveryInitiative, the University of Tokyo, or Prestwick Chemical Library.

Item [16-2] The screening method according to any one of items [12] to[15], wherein the screening method is a high throughput screeningmethod.

(Medical Use)

Item [17] A pharmaceutical composition for preventing or treating alipid peroxidation reaction-induced disease or inhibiting progression ofthe lipid peroxidation reaction-induced disease in a subject, comprisingan effective amount of at least one compound selected from the groupconsisting of a group:

apomorphine ((R)-(−)-apomorphine hydrochloride), eseroline((−)-eseroline fumarate), ethoxyquine(6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline), methyldopa (methyldopasesquihydrate), olanzapine(2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine,methyl 3-amino-4-(phenylamino)benzoate (compound 52), methyl3-amino-4-((4-methoxyphenyl)amino)benzoate (compound 52-1), methyl3-amino-4-((3-methoxyphenyl)amino)benzoate (compound 52-3), methyl3-amino-4-(benzylamino)benzoate (compound 52-4), methyl3-amino-4-((1-phenylethyl)amino)benzoate (compound 52-5),1-(4-(trifluoromethoxy)phenyl)indolin-5-amine (compound 78),1-(3,5-dimethylphenyl)-1H-indol-6-amine (compound 78-3),1-(3,5-dimethylphenyl)indolin-6-amine (compound 78-4),1-(4-methoxyphenyl)-1H-indol-6-amine (compound 78-5),1-(4-(methylthio)phenyl)-1H-indol-6-amine (compound 78-6),1-(4-(trifluoromethoxy)phenyl)-1H-indol-5-amine (compound 78-8).

Item [18] The pharmaceutical composition according to item [17], whereinthe disease is selected from the group consisting of Alzheimer-typedementia, chronic kidney diseases, diabetic neuropathy, liver disorder,age-related macular degeneration, postischemic brain disorder, vasculardementia, arteriosclerosis, Parkinson's disease, multiple sclerosis,cancer, asthma, hypertension, cardiovascular diseases, and age-relatedeye disease.

Item [19] The pharmaceutical composition according to item [17], whereinthe disease is age-related macular degenerative disease.

Item [20] A method for preventing or treating a lipid peroxidationreaction-induced disease or inhibiting progression of the lipidperoxidation reaction-induced disease, with at least one compoundselected from the group consisting of the group described in item [17].

Item [21] Use of at least one compound selected from the groupconsisting of the group described in item [17] in the manufacture of amedicament for preventing or treating a lipid peroxidationreaction-induced disease or inhibiting progression of the lipidperoxidation reaction-induced disease.

Item [22] Use of at least one compound selected from the groupconsisting of the group described in item [17], for preventing ortreating a lipid peroxidation reaction-induced disease or inhibitingprogression of the lipid peroxidation reaction-induced disease.

Effects of Invention

The assay kit of the present invention and the screening method usingthe assay kit enable to easily explore a compound having a lipidperoxidation inhibitory effect. Furthermore, the compounds found by thescreening method of the present invention are useful for treatment oflipid peroxidation reaction-induced diseases (for example, age-relatedmacular degeneration), or the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a chart showing the relationship between lipid peroxidationreaction and diseases.

FIG. 2 is a chart showing the result of a test for evaluating theresponsiveness of profluorescent nitroxide probes to lipid peroxidationreaction. Liposomes (2.5 mg/mL EggPC, 0.1 mg DCP), 5.0 μM profluorescentnitroxide, and 20 mM AAPH were mixed, and after 40 minutes, thefluorescence intensity (NBD-TEMPO: λ_(Ex)/λ_(Em)=470/530 nm,Dansyl-TEMPO: λ_(Ex)/λ_(Em)=300/500 nm) was measured (n=3, mean+S.D.,**p<0.01 v.s. ctrl(−)).

FIG. 3A-FIG. 3B is a chart showing the result of a test for evaluatingthe reactivity between profluorescent nitroxide probes and variousreductants. Liposomes (2.5 mg/mL EggPC, 0.1 mg DCP), 5.0 μMprofluorescent nitroxide, and 50 μM antioxidant were mixed, and after 40minutes, the fluorescence intensity (λ_(Ex)/λ_(Em)=470/530 nm) wasmeasured. The ratio of the fluorescence intensity is a value obtained bydividing the fluorescence intensity of the probe after reaction with thevarious antioxidants by the fluorescence intensity of ctrl(−). FIG. 3AChemical structure of the antioxidant used in this study; FIG. 3B Ratioof fluorescence intensity due to the reaction between the probe and theantioxidant (n=3, mean+S.D., *p<0.05, **p<0.01 v.s. ctrl(−)).

FIG. 4 is a chart showing the result of a test for evaluating thereactivity between profluorescent nitroxide probes and various oxidants.5.0 μM profluorescent nitroxide and various oxidants were mixed, andafter 30 minutes, the fluorescence intensity (λ_(Ex)/λ_(Em)=470/530 nm)was measured. The ratio of fluorescence intensity is a value obtained bydividing the fluorescence intensity of the probe after reaction with thevarious oxidants by the fluorescence intensity of ctrl(−). As theoxidants, 0.5 mM hydrogen peroxide, 0.5 mM hypochlorous acid, 0.5 mMpotassium oxide, 0.5 mM hydrogen peroxide and 5.0 μM FeSO₄ were used,and liposomes (2.5 mg/mL EggPC, 0.1 mg DCP) and 10 mM AAPH were used(n=3, mean+S.D., **p<0.01 v.s. ctrl(−)).

FIGS. 5A-FIG. 5D is charts showing the results of a test for evaluatingthe reaction initiator concentration-dependent lipid peroxidationreaction in artificial lipid membranes in an AAPH system. Liposomes (2.5mg/mL EggPC, 0.1 mg DCP), 5.0 μM NBD-TEEPO, and AAPH were mixed, andafter 40 minutes, the fluorescence intensity (λ_(Ex)/λ_(Em)=470/530 nm)was measured. FIG. 5A Stimulation concentration-dependency (AAPH 0-20mM); FIG. 5B Antioxidant concentration-dependency (AAPH 20 mM, AsA 0-20μM) of NBD-TEEPO fluorescence responsiveness. Comparison of FIG. 5Clipid peroxidation reaction evaluation method using NBD-TEEPO (AAPH 20mM, antioxidant 10 μM) and FIG. 5D TBARS assay (AAPH 20 mM, antioxidant10 μM) (n=3, mean+S.D., **p<0.01 v.s. ctrl(−), *p<0.05, **p<0.01 v.s.ctrl(+)).

FIGS. 6A-FIG. 6D is charts showing the results of a test for evaluatingthe reaction initiator concentration-dependent lipid peroxidationreaction in artificial lipid membranes in an Fe²⁺ system. Liposomes (2.5mg/mL EggPC, 0.1 mg DCP), 5.0 μM NBD-TEEPO and FeSO₄ were mixed, andafter 60 minutes, the fluorescence intensity (λ_(Ex)/λ_(Em)=470/530 nm)was measured. FIG. 6A Stimulation concentration-dependency (FeSO₄ 0-2mM); FIG. 6B Antioxidant concentration-dependency (FeSO₄ 1 mM, Eda 0-20μM) of NBD-TEEPO fluorescence responsiveness. Comparison of FIG. 6Clipid peroxidation reaction evaluation method using NBD-TEEPO (FeSO₄ 1mM, antioxidant 10 μM) and FIG. 6D TBARS assay (FeSO₄ 1 mM, antioxidant10 μM) (n=3, mean+S.D., **p<0.01 v.s. ctrl(−), ^(##)p<0.05, ^(##)p<0.01v.s. ctrl(+)).

FIG. 7A-FIG. 7E is charts showing the results of a test for evaluatingintracellular lipid peroxidation reaction in cultured cell systems. To1.0×10⁴HepG2 cells, 5.0 μM NBD-TEEPO and 50 μM various oxidants andantioxidants were added, and changes of the fluorescence intensity(λ_(Ex)/λ_(Em)=470/530 nm) were measured. FIG. 7A Fluorescence intensityof NBD-TEEPO at 45 minutes after addition of AA (0 to 200 μM); FIG. 7BFluorescence intensity of NBD-TEEPO at 45 minutes after addition of 200μM AA and 50 μM antioxidant; FIG. 7C Fluorescence intensity of NBD-TEEPOat 45 minutes after addition of tBHP (0 to 300 μM); FIG. 7D Fluorescenceintensity of NBD-TEEPO at 45 minutes after addition of 300 μM tBHP and50 antioxidant; FIG. 7E S/B ratios, CV values, and Z′-factors (AA-addedsystem: 45 minutes after addition of 200 μM AA; tBHP-added system: 45minutes after addition of 300 μM tBHP) (n=3, mean+S.D., **p<0.01 v.s.ctrl(−), ^(#)p<0.05, ^(##)p<0.01 v.s. ctrl(+)).

FIGS. 8A-FIG. 8E is charts showing the results of a test for evaluatingchanges in lipid peroxidation-induced cell viability in cultured cellsystems using an MTT assay. To 1.0×10⁴ HepG2 cells were added 50 μMvarious oxidants and antioxidants, and the cell viability after 24 hourswas measured by an MTT assay (λ_(max)=570 nm). The cell viability wascalculated with a 0 μM oxidation stimulus and 0 μM antioxidant as 100%.FIG. 8A Cell viability at 24 hours after addition of AA (0 to 100 μM);FIG. 8B Cell viability at 24 hours after addition of 100 μM AA and 50 μMantioxidant; FIG. 8C Cell viability at 24 hours after addition of tBHP(0 to 100 μM); FIG. 8D Cell viability at 24 hours after addition of 100μM tBHP and 50 μM antioxidant; (e) S/B ratios, CV values, and Z′-factors(AA-added system: 24 hours after addition of 100 μM AA, tBHP-addedsystem: 24 hours after addition of 100 μM tBHP). (n=3, mean+S.D.,**p<0.01 v.s. ctrl(−), ^(#)p<0.05, ^(##)p<0.01 v.s. ctrl(+)).

FIGS. 9A-FIG. 9B is drawings showing schematic diagrams of screeningmethods.

FIG. 10 is a chart showing the result of primary screening using CoreLibrary of Drug Discovery Initiative, the University of Tokyo, in anAAPH system. Liposomes (2.5 mg/mL EggPC, 0.1 mg DCP), 5.0 μM NBD-TEEPO,and 20 mM AAPH were mixed, and the fluorescence intensity(λ_(Ex)/λ_(Em)=470/530 nm) was measured. The activity values of eachcompound at 40 minutes after the reaction are shown.

FIG. 11 is a chart showing the result of AUC evaluation in an Fe²⁺system. Liposomes (2.5 mg/mL EggPC, 0.1 mg DCP), 5.0 μM NBD-TEEPO and1.0 mM FeSO₄ were mixed, and the fluorescence intensity(λ_(Ex)/λ_(Em)=470/530 nm) was measured over time for 180 minutes. Thearea under the curve (AUC) was calculated from the obtained curve.

FIG. 12 is a chart showing the result of primary screening using CoreLibrary of Drug Discovery Initiative, the University of Tokyo, in anFe²⁺ system. Liposomes (2.5 mg/mL EggPC, 0.1 mg DCP), 5.0 μM NBD-TEEPOand 1.0 mM FeSO₄ were mixed, and the fluorescence intensity(λ_(Ex)/λ_(Em)=470/530 nm) was measured. The activity values of eachcompound at 180 minutes after the reaction are shown.

FIGS. 13A-FIG. 13D is charts showing the results of secondary screeningusing Core Library of Drug Discovery Initiative, the University ofTokyo. To 1.0×10⁴ HepG2 cells, 5.0 μM NBD-TEEPO, 200 μM AA or 300 μMtBHP, and 50 μM compound were added, and changes of the fluorescenceintensity (λ_(Ex)/λ_(Em)=470/530 nm) were measured. The dotted lines inFIG. 13A and FIG. 13B represent the activity values of Edaravone. FIG.13A Activity value of each compound at 45 minutes after addition of AA;FIG. 13B Activity value of each compound at 60 minutes after addition oftBHP; FIG. 13C Plot of activity values in the AA-added system andtBHP-added system; FIG. 13D) Enlarged view of the part in which theactivity value in the AA-added system is 30% or more and the activityvalue in the tBHP-added system is 50% or more in FIG. 13C.

FIGS. 14A-FIG. 14D is charts showing the results of secondary screeningusing Core Library of Drug Discovery Initiative, the University ofTokyo, according to an MTT assay method. To 1.0×10⁴HepG2 cells, 100 μMAA or tBHP and 50 μM compound were added, and the cell viability after24 hours was measured by an MTT assay. The dotted lines in FIG. 14A andFIG. 14B represent the activity value of Edaravone. FIG. 14A Activityvalue of each compound at 24 hours after addition of AA; FIG. 14BActivity value of each compound at 24 hours after addition of tBHP; FIG.14C Plot of activity values in the AA-added system and tBHP-addedsystem; FIG. 14D Enlarged view of the part in which the activity valuein the AA-added system is 30% or more and the activity value in thetBHP-added system is 50% or more in FIG. 14C.

FIGS. 15A-FIG. 15D is charts showing the results of tertiary screeningusing Core Library of Drug Discovery Initiative, the University ofTokyo. To 1.0×10⁴HepG2 cells, 5.0 μM NBD-TEEPO, 200 μM AA or 300 μM tBHPand 50 μM compound were added, and changes of the fluorescence intensity(λ_(Ex)/λ_(Em)=470/530 nm) were measured (white bar: activity value ofthe compound of original structure, black bar: activity value of astructural analog, dotted line: activity value of Edaravone). FIG. 15AActivity value of each compound at 45 minutes after addition of AA; FIG.15B Activity value of each compound at 60 minutes after addition oftBHP; FIG. 15C Plot of activity values in the AA-added system andtBHP-added system; FIG. 15 d Enlarged view of the part in which theactivity values in the AA-added system and the activity value in thetBHP-added system are 50% or more in FIG. 15C.

FIGS. 16A-FIG. 16D is charts showing the results of tertiary screeningusing Core Library of Drug Discovery Initiative, the University ofTokyo, according to an MTT assay method. To 1.0×10⁴ HepG2 cells, 100 μMAA or tBHP and 50 μM compound were added, and the cell viability after24 hours was measured by an MTT assay (white bar: activity value of thecompound of original structure, black bar: activity value of astructural analog compound, dotted line: activity value of Edaravone).FIG. 16A Activity value of each compound at 24 hours after addition ofAA; FIG. 16B Activity value of each compound at 24 hours after additionof tBHP; FIG. 16C Plot of activity values in the AA-added system andtBHP-added system; FIG. 16D Enlarged view of the part in which theactivity value in the AA-added system is 0% or more and the activityvalue in the tBHP-added system is 40% or more in FIG. 16C.

FIG. 17 is a chart showing the result of a test for cytotoxicityevaluation at tertiary screening using Core Library of Drug DiscoveryInitiative, the University of Tokyo. To 1.0×10⁴ HepG2 cells, 50 μMcompound were added, and cell viability after 72 hours was measured byan MTT assay. The dotted line represents the cell viability at 72 hoursafter incubation with Edaravone.

FIGS. 18A-FIG. 18B is charts showing the results of primary screeningusing Prestwick Chemical Library. Liposomes (2.5 mg/mL EggPC, 0.1 mgDCP), 5.0 μM NBD-TEEPO and a reaction initiator were mixed and thefluorescence intensity changes (λ_(Ex)/λ_(Em)=470/530 nm) were measured.FIG. 18A Activity value of each compound at 40 minutes after startingmeasurement in an AAPH system; FIG. 18B Activity value of each compoundat 180 minutes after starting measurement in an Fe²⁺ system.

FIGS. 19A-FIG. 19C is charts showing the results of primary screeningusing Prestwick Chemical Library and the relationship between candidatecompounds and target disease areas. FIG. 19A Plot of activity values ofthe AAPH system and the Fe²⁺-added system at primary screening; FIG. 19BTarget disease areas of the top 16 compounds; FIG. 19C) Enlarged view ofthe part in which the activity value in the AAPH system is 0.7 or oreand the activity value in the Fe²⁺-added system is 0.8 or more in FIG.19A.

FIGS. 20A-FIG. 20B is charts showing a method for producing age-relatedmacular degeneration (AMD) model mice. FIG. 20A AMD model mouseproduction schedule; FIG. 20B ONL measurement method in which ONLthickness was measured over 27 points (A-center-Z) every 180 μm (left:observation field of 4 times, right: observation field of 60 times).

FIGS. 21A-FIG. 21C is charts showing the results of evaluating thethickness of an outer nuclear layer (ONL). FIG. 21A ONL bright fieldimage (observation magnification: 60 times); FIG. 21B ONL thickness(model compound: compound W). The left represents the inferiorhemisphere of eyeball, and the right represents the superior hemisphereof eyeball. FIG. 21C Mean thickness of the ONL at points Q to T (n=3-5,mean+S.D., *p<0.05, **p<0.01 v.s. ctrl(−), ^(#)p<0.05, ^(##)p<0.01 v. s.ctrl(+)).

FIG. 22 is a table summarizing main action/action points, half-lethaldose (LD₅₀) and animal administration examples for the activityindicator compound and candidate compounds. The main action/actionpoints, LD₅₀ and animal administration examples were shown for Edaravoneand the five compounds used in this study. Here, oral represents oraladministration, s.c. represents subcutaneous injection, and i.v.represents intravenous injection.

DESCRIPTION OF EMBODIMENTS

(Assay Method and Assay Kit)

The present inventors provide assay methods and assay kits fordeveloping screening methods (for example, high-throughput screeningmethods) capable of testing and evaluating a large number of compoundsat once for the purpose of exploring lipid peroxidation inhibitors.

The present invention provides an assay kit for detecting lipidperoxidation inhibitory activity of a test compound, comprising

a compound represented by formula (I):

a liposome, andat least one compound selected from the group consisting of2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ionsource materialin a buffer.

Examples of the liposome used in the assay kit of the present inventioninclude a liposome produced from egg yolk-derived phosphatidylcholine(egg yolk-derived phosphatidylcholine (Egg PC)) and dihexadecyl hydrogenphosphate (DCP) as a lipid source.

Examples of the radical reaction initiator include at least one compoundselected from the group consisting of either 2,2′-azobis(2-aminopropane)dihydrochloride (hereinafter referred to as “AAPH”) or divalent iron ionsource materials (for example, FeSO₄).

The assay kit of the present invention includes a solution of buffer(for example, phosphate buffer).

Profluorescent Nitroxide Compound

In the assay method and the assay kit of the present invention, aprofluorescent nitroxide compound represented by formula (I) is used asa lipid radical scavenger. The profluorescent nitroxide compound isdescribed in the literature by the present inventors (for example,Japanese Patent Application No. 2017-090739).

Here, nitroxide (NO.) refers to a stable radical having paramagnetism.The nitroxide has a property of attenuating fluorescence due tophoto-induced electron transfer accompanied by a charge separation stateand intersystem crossing by electron-spin exchange. Profluorescentnitroxide in which a fluorescent chromophore is covalently bonded tonitroxide is in an intramolecular quenching state. However, it has beenconfirmed that when nitroxide reacts with free radicals and loses theparamagnetism, it is in a fluorescent emission state. Namely, theprofluorescent nitroxide is useful as a probe for detecting lipidradical scavenging by fluorescence observation, and enables to evaluatethe lipid radical scavenging by measuring fluorescence intensity.

Most of the lipid molecules to be detected are present in biologicalmembranes and form a hydrophobic environment. Thus, an environmentallyresponsive fluorescent chromophore that emits selectively highfluorescence in hydrophobic environments while the fluorescence isattenuated in hydrophilic environments is optimal. Examples of thefluorescent chromophore include fluorescent chromophores widely used inthe lipid field such as biological membrane phase transition andmembrane fusion or intracellular lipid metabolism, for example,nitrobenzofurazan (hereinafter referred to as “NBD”)) and5-(dimethylamino)naphthalene-1-sulfonyl chloride) (hereinafter referredto as “Dansyl”).

Examples of a probe molecule that is an α-position substituent of thefluorescent nitroxide compound include2,2,6,6-tetramethylpiperidin-1-oxyl (hereinafter referred to as“TEMPO”), 2,2,6,6-tetraethylpiperidin-1-oxyl (hereinafter referred to as“TEEPO”), and 2,2,6-trimethyl-6-pentyl-piperidin-1-oxyl (hereinafterreferred to as “Pen”).

For selecting a profluorescent nitroxide compound suitable for the assaymethods and assay kits of the present invention, each of the fluorophoremolecule and probe molecule are optimized. The optimization of thefluorophore molecule is performed by testing the responsiveness to lipidperoxidation reaction.

The optimization of the probe molecule that is an α-position substituentof the fluorescent nitroxide compound is performed by a test forevaluating the reactivity with reductants and a test for evaluating thereactivity with oxidants. In the assay method of the present invention,inhibition is evaluated by inhibition of lipid peroxidation reaction bya reductant (i.e., an antioxidant), that is, whether or not an increasein the fluorescence intensity due to the lipid peroxidation reactiononce occurred can be reduced by the antioxidant (for example, a testcompound). In this case, a direct reaction of the probe molecule withthe antioxidant to cause an increase of fluorescence intensity leads todetection of false negatives. Thus, first, the reactivity of the probemolecule with antioxidants is examined.

Then, the responsiveness of the probe molecule to the lipid peroxidationreaction is examined by a test for evaluating the reactivity of theprobe molecule on a profluorescent nitroxide compound with oxidants.Here, reactive oxygen species •OH is generated by hydrogen peroxide andFe²⁺. Lipid peroxidation reaction is caused by liposomes and AAPH.

From the results of the test, the NBD-TEEPO compound that exhibits thehighest reduction resistance and high responsiveness to the lipidperoxidation reaction is selected as the profluorescent nitroxidecompound.

Establishment of Assay Method and Assay Kit

The present invention provides an assay method and an assay kit usingthe NBD-TEEPO compound represented by the formula (I). The assay methodand the assay kit can be applied to a screening method.

Cell-Free Based Assay Methods

The present invention provides a cell-free based assay method fordetecting lipid peroxidation inhibitory activity, using an NBD-TEEPOcompound represented by formula (I).

Similar to the above method for optimization of profluorescent nitroxidecompounds, liposomes are used as lipid, and AAPH and FeSO₄ are used asradical reaction initiators. In both AAPH and Fe²⁺ systems, thefluorescence intensity of the probe increases concentration-dependently.In the AAPH system, when a water-soluble antioxidant (for example,ascorbic acid (AsA)) is used, the increase in fluorescence is inhibitedconcentration-dependently, while in the Fe²⁺ system, when a lipophilicantioxidant (for example, Edaravone (eda)) is used, the increase influorescence is inhibited concentration-dependently. In both assaymethods, by using a plurality of known antioxidants, it can be foundthat the assay method of the present invention is an assay method thatcan evaluate the lipid peroxidation reaction and the lipid peroxidationreaction inhibitory effect of antioxidants.

Further, when comparing the result of the assay method of the presentinvention with the result of 2-thiobarbituric acid reactive substance(hereinafter referred to as “TBARS”) method, which is known as a methodto measure the lipid peroxidation inhibitory effect, using the sameknown antioxidants, similar results are obtained. It can be found thatthe assay method of the present invention is an assay method that canevaluate the lipid peroxidation reaction and the inhibitory effect ofantioxidants to the reaction. Moreover, the assay method of the presentinvention does not require a complicated procedure which is required inthe TBARS method.

In one embodiment, a specific assay method includes the following steps:

i) preparing a buffer containing a compound represented by formula (I)and liposomes;

ii) adding at least one compound selected from the group consisting of2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ionsource material;

iii) adding a test compound;

iv) measuring fluorescence; and

v) determining an activity value of the test compound from the result ofmeasuring the fluorescence.

In one embodiment, the cell-free based assay kit of the presentinvention includes a combination of:

an assay kit in which the reaction initiator is2,2′-azobis(2-aminopropane) dihydrochloride; and

an assay kit in which the reaction initiator is iron(II) sulfate.

Composition of Cell-Free Based Assay Kit

In the cell-free based assay kit of the present invention, the compoundrepresented by the formula (I) has a concentration of 1.0 to 20.0 μM(for example, 5.0 to 20.0 μM, typically 5.0 μM);

-   -   the liposome is prepared from egg yolk-derived        phosphatidylcholine and dihexadecyl hydrogen phosphate, and the        egg yolk-derived phosphatidylcholine has a concentration of 5.0        to 10.0 mg/mL (for example, 2.5 mg/mL) and the dihexadecyl        hydrogen phosphate has a concentration of 0.01 to 1.0 mg/mL (for        example, 0.1 mg/mL),        the test compound has a concentration of 5 to 100 μM (for        example, 10 μM);        the 2,2′-azobis(2-aminopropane) dihydrochloride has a        concentration of 5 to 50 mM (for example, 20 mM), and        a divalent iron ion source material (for example, FeSO₄) has a        concentration of 0.5 to 50 mM (for example, 1 mM).

Furthermore, since the cell-free based assay method of the presentinvention has sufficient values in the indicators representing thequality of screening system (for example, S/B ratio, CV value,Z′-factor), it can be applied to a screening method.

Cell-Based Assay Method

The present invention provides, in addition to the cell-free based assaymethod described above, a cell-based assay method for detecting lipidperoxidation inhibitory activity using an NBD-TEEPO compound representedby formula (I).

The cell-based assay method is performed using cultured cells (forexample, human hepatoma-derived HepG2 cells) instead of liposomes usedin the cell-free based assay, and using arachidonic acid (hereinafterreferred to as “AA”) and tert-butyl hydroperoxide (hereinafter referredto as “tBHP”) instead of AAPH and a divalent iron ion source material asradical reaction initiators.

In one embodiment, a specific assay method includes the following steps:

i) preparing a buffer containing a compound represented by formula (I)and a cultured cell;

ii) adding at least one compound selected from the group consisting ofarachidonic acid and tert-butyl hydroperoxide,

iii) adding a test compound;

iv) measuring fluorescence; and

v) determining an activity value of the test compound from the result ofmeasuring the fluorescence.

In one embodiment, the cell-based assay kit of the present inventionincludes a combination of:

an assay kit in which a reaction initiator is arachidonic acid; and

an assay kit in which a reaction initiator is tert-butyl hydroperoxide.

Composition of Cell-Based Assay Kit

In the cell-based assay kit of the present invention,

the compound represented by formula (I) has a concentration of 1.0 to20.0 μM (for example, 5.0 μM),

the cultured cell has a concentration of 1×10⁴ to 1×10⁵ cells (forexample, 1×10⁴ cells),

the test compound has a concentration of 5 to 500 μM (for example, 50μM);

the arachidonic acid has a concentration of 100 to 400 μM (for example,200 μM), and

the tert-butyl hydroperoxide has a concentration of 100 to 400 μM (forexample, 300 μM).

Furthermore, since the cell-based assay method of the present inventionhas sufficient values in the indicators representing the quality ofscreening system (for example, S/B ratio, CV value, Z′-factor), it canbe applied to a screening method.

The assay kit of the present invention may include a package insertshowing an activity value of a compound having lipid peroxidationinhibitory activity. Evaluation of lipid peroxidation inhibitoryactivity of a test compound can be performed by comparing the activityvalue of lipid peroxidation inhibition of the test compound obtainedusing the assay kit or assay method of the present invention with theactivity value of the indicator compound shown in the package insert.

In one embodiment, the assay method of the present invention includes acombination of at least two or more assay methods selected from thegroup consisting of:

a cell-free based assay method in which the reaction initiator is2,2′-azobis(2-aminopropane) dihydrochloride;

a cell-free based assay method in which the reaction initiator isiron(II) sulfate;

a cell-based assay method in which the reaction initiator is arachidonicacid; and

a cell-based assay method in which the reaction initiator is tert-butylhydroperoxide.

In one embodiment, the assay kit of the present invention includes acombination of at least two or more assay kits selected from the groupconsisting of:

a cell-free based assay kit in which the reaction initiator is2,2′-azobis(2-aminopropane) dihydrochloride;

a cell-free based assay kit in which the reaction initiator is iron(II)sulfate;

a cell-based assay kit in which the reaction initiator is arachidonicacid; and

a cell-based assay kit in which the reaction initiator is tert-butylhydroperoxide.

Lipid peroxidation-induced cell death is caused in the process in whichlipid peroxidation reaction promotes development and progression ofdiseases (Reference: Uchida K., Prog. Lipid Res., 2003, 42(4), 318-43).A method using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (hereinafter referred to as “MTT”) is used for evaluation ofcell viability. It is also known that the MTT method can be carried outby plate assay.

In one embodiment, the assay method of the present invention includes anassay method according to an MTT method, including the following steps:

i) performing an assay method according to an MTT method using a culturemedium containing a cultured cell, a test compound and arachidonic acid,and selecting a compound having high cell viability; and/or

ii) performing an assay method according to an MTT method using aculture medium containing a cultured cell, a test compound andtert-butyl hydroperoxide, and selecting a compound having high cellviability.

The assay method and the assay kit of the present invention may includethe assay method and kit according to an MTT method in combination withthe cell-free based and/or cell-based assay method and the assay kit ofthe present invention.

Since this MTT method has sufficient values in the indicatorsrepresenting the quality of screening system (for example, S/B ratio, CVvalue, Z′-factor), it can be applied to a screening method.

Any assay method of the present invention can also be performed with amicrowell plate. Examples of the microwell plate include a porous plate(for example, a 96-well plate and a 384-well plate), but are not limitedto these. For example, commercially available microwell plates can alsobe used.

In one embodiment, a measurement by the assay method of the presentinvention using a microwell plate includes the following steps:

i) dispensing a solution of a test compound into the microwell plate;

ii) dispensing a solution containing a compound represented by formula(I) and a liposome or a cultured cell into each well;

iii) when using the liposome, dispensing a solution containing at leastone compound selected from the group consisting of2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ionsource material into the each well, and when using the cultured cell,dispensing a solution containing at least one compound selected from thegroup consisting of arachidonic acid and tert-butyl hydroperoxide intothe each well; andiv) measuring fluorescence with a microplate reader.(Screening Method)

The present invention provides a screening method including a screeningstep using the assay method of the present invention for a compoundlibrary to explore compounds having lipid peroxidation inhibitoryactivity.

Here, the compound library may or may not be a known one. Examples ofthe known compound library include compound libraries that collectcompounds that have already been approved as food (for example, by U.S.Food and Drug Administration (FDA)) or as pharmaceutical (for example,by European Agency for the Evaluation of Medicinal Products (EMEA)) (forexample, the PRESTWICK CHEMICAL library, which is the collection ofcompounds with expired patent term), and compound libraries that collectcompounds that have not yet been approved as food or pharmaceutical (forexample, Core Library in the General Library of Drug DiscoveryInitiative, the University of Tokyo,). A schematic diagram of thescreening method of the present invention is shown in FIGS. 9A-FIG. 9B.

Primary Screening

In the screening by the liposome-AAPH system assay method, compoundshaving a radical scavenging ability, ranging from highly water-solubleantioxidants that inhibit water-soluble AAPH-derived radical species tohighly lipid-soluble antioxidants that inhibit lipid peroxidation chainreactions can be detected. In addition, in the screening by theliposome-Fe²⁺ system assay method, more lipophilic compounds among thecompounds with radical scavenging ability are more easily detected.However, iron chelating agents without radical scavenging ability may bedetected. Thus, in this primary screening, first, candidate compoundshaving radical scavenging ability are broadly selected from testcompounds by AAPH system screening, then, from the compounds narroweddown by the liposome-AAPH system screening, candidate lipid-solublecompounds are further narrowed down by liposome-Fe²⁺ system screening.

The screening method using the assay method of the present inventionincludes, as the primary screening, a screening by the cell-free basedassay method using liposomes.

The primary screening method of the present invention includes thefollowing method: a screening method for selecting a candidate compoundhaving high lipid peroxidation inhibitory activity, including:

i) selecting a test compound from a compound library;

ii) performing a screening using the test compound by a cell-free assaymethod using 2,2′-azobis(2-aminopropane) dihydrochloride (hereinafterreferred to as “AAPH”)(hereinafter, this assay method referred to as“liposome-AAPH system”), and selecting a compound having a high activityvalue; andiii) then, performing a screening using the compound having a highactivity value in ii) by a cell-free assay method using a divalent ironion source material (hereinafter, this assay method referred to as“liposome-Fe²⁺ system”), and selecting a compound having a high activityvalue.

From the fluorescence intensity measured in the screening of the aboveii), the activity value of each test compound is calculated based on thefollowing expression:Activityvalue=1−(Flu_(Sample)−Flu_(Background))/(Flu_(Control)−Flu_(Background))Flu_(Sample): Fluorescence intensity for with AAPH and with eachcompound (n=1)Flu_(Background): Fluorescence intensity for without AAPHFlu_(Control): Fluorescence intensity for with AAPH and without eachcompound

From the fluorescence intensity measured in the screening of the aboveiii), the activity value of each test compound is calculated based onthe following expression:Activity value=1−(AUC_(Sample)/AUC_(Control))AUC_(Sample): Area under the curve calculated from the fluorescenceintensity for with Fe²⁺ and with each compound (n=1)AUC_(Control): Area under the curve calculated from the fluorescenceintensity for with Fe²⁺ and without each compound.

Narrowing of candidate compounds is performed in comparison with theactivity value of known compounds which have been known to have highlipid peroxidation inhibitory activity (hereinafter referred to as“activity indicator compounds”). Examples of the activity indicatorcompound include Edaravone,4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol) and(−)-epicatechin.

Secondary Screening

When using a compound library that collects compounds unapproved as foodor pharmaceutical (for example, Core Library in the General Library ofDrug Discovery Initiative, the University of Tokyo,) as the compoundlibrary, the screening using the assay method of the present inventionmay employ a screening by the cell-based assay method of the presentinvention using a cultured cell (for example, human hepatoma-derivedHepG2 cell), as higher-order screening (for example, secondaryscreening), for the purpose of investigating lipid peroxidationinhibitory activity and cell death inhibitory activity in a cell system.On the other hand, when using a compound library that collects compoundsalready approved as food or pharmaceutical, there is no need toinvestigate lipid peroxidation inhibitory activity and cell deathinhibitory activity in the cell system, thus the secondary screening maybe omitted accordingly.

In the screening for investigating lipid peroxidation inhibitoryactivity in the cell system, a screening is performed by the cell-basedassay method of the present invention, and activity values aredetermined based on the results of measuring the fluorescence intensity.Then, candidate compounds are narrowed down in comparison with theactivity value of the activity indicator compound.

The secondary screening method of the present invention includes amethod including:

i) performing a screening by a cell-based assay method using arachidonicacid, and selecting a compound having a high activity value;

ii) performing a cell-based assay method using tert-butyl hydroperoxide,and selecting a compound having a high activity value; and

iii) selecting a compound having high activity values in both of theassay methods of i) and ii).

In the screening by the cell-based assay method, activity values weredetermined from the values of fluorescence intensity measured in eachscreening using AA or tBHP as a reaction initiator, based on thefollowing expression:Activityvalue=100(AUC_(Sample)−AUC_(Background))/(AUC_(Control)−AUC_(Background))AUC_(Sample): Area under the curve calculated from the fluorescenceintensity for with reaction initiator and with each compound (n=1)AUC_(Control): Area under the curve calculated from fluorescenceintensity for with reaction initiator and without each compoundAUC_(Background): Area under the curve calculated from fluorescenceintensity for without reaction initiator and without each compound

Then, the candidate compounds are narrowed down in comparison with thevalue of the activity indicator compound. The activity values obtainedin each screening using AA or tBHP are plotted. Candidate compoundshaving high activity values for lipid peroxidation inhibition in both ofthe screenings are selected.

Moreover, compounds which have high lipid peroxidation-induced celldeath inhibitory activity are explored.

As a cell-based screening for examining the cell death inhibitoryactivity, an assay using human hepatoma-derived HepG2 cells and AA andtBHP as radical initiators is performed, and the cell viability ismeasured. The activity value is determined from the cell viability valuebased on the following expression:Activityvalue=100(Viability_(Sample)−Viability_(Control(+)))/(Viability_(Control(−))−Viability_(Control(+)))Viability_(Sample): Cell viability for with reaction initiator and witheach compound (n=1)Viability_(Control(+)): Cell viability for with reaction initiator andwith each compoundViability_(Control(−)): Cell viability for without reaction initiatorand without each compound

Then, the candidate compounds are narrowed down in comparison with thevalue of the activity indicator compound. The activity values obtainedin each assay system using AA or tBHP are plotted. Candidate compoundshaving high activity values for cell death inhibition (cell deathsuppression) in both of the assay systems are selected.

In one embodiment, the secondary screening method of the presentinvention further includes a method including:

i) performing a screening by an assay method according to an MTT methodusing a culture medium containing a cultured cell, a test compound, andarachidonic acid, and selecting a compound having high cell viability;

ii) performing a screening by an assay method according to an MTT methodusing a culture medium containing a cultured cell, a test compound, andtert-butyl hydroperoxide, and selecting a compound having high cellviability; and

iii) selecting a candidate compound having high cell viability in bothof the assay methods of i) and ii).

Tertiary Screening

The compound library to be used (for example, Core Library of DrugDiscovery Initiative, the University of Tokyo,) may include, forexample, structural analogs related to candidate compounds that havehigh activity values in the above-described low-order screening assays(for example, secondary screening). Thus, by performing higher-orderscreening (for example, tertiary screening) of such structural analogs,optionally, together with the candidate compound selected in thelow-order screening assays for lipid peroxidation inhibitory activity,candidate compounds are selected.

Moreover, these candidate compounds are further screened by an assaymethod for examining cell death inhibitory activity.

Finally, a candidate compound is chosen entirely taking into account theresult of the final high-order screening (for example, tertiaryscreening) and the result of the screening for cell death inhibitoryactivity.

In one embodiment, the screening method of the present inventionincludes:

i) selecting a structural analog of a compound selected by a screeningmethod by the cell-based assay of the present invention from a compoundlibrary;

ii) performing the screening method by the cell-based assay of thepresent invention for the compound selected in i) and optionally theoriginal compound of the selection, and selecting a compound having ahigh activity value;

iii) performing the screening method by the cell-based assay of thepresent invention according to an MTT method for the compound selectedin i) and optionally the original compound of the selection, andselecting a compound having high cell viability;

iv) selecting a candidate compound having a high activity value and highcell viability in the screening methods of ii) and iii) and

v) performing a screening by an assay method using a culture mediumcontaining a cultured cell for the compound selected in i) andoptionally a compound selected by screening by the cell-based assay orscreening by the cell assay according to an MTT method, and selecting acandidate compound having a high cell viability; andvi) selecting a candidate compound from the compound selected in iv) andthe compound selected in v).

The screening method of the present invention can be used as ahigh-throughput screening method.

(Utilization of Screening Results)

Diseases involving lipid radicals in lipid peroxidation reactions covera wide range of disease areas. Thus, candidate compounds can be narroweddown for each target disease in consideration of other factors based onthe knowledge of the action mechanism. For example, when targeting adisease requiring permeability to the blood-brain barrier, candidatecompounds may be further narrowed down considering lipid solubility.Specifically, when targeting cerebral infarction and retinal diseases(for example, age-related macular degeneration), compounds that arepermeable to the blood brain barrier are advantageous, while whentargeting hepatoma and arteriosclerosis, permeability to the blood brainbarrier is not required.

(Medical Use)

As used herein, “treating” or “preventing” a disease caused by lipidperoxidation reaction encompasses one or more of the followings: (1)removing the disease; (2) reducing or minimizing the severity of thedisease; (3) delaying the progression or onset of the disease; and (4)reducing, minimizing, or eliminating the occurrence or frequency of thedisease.

As used herein, “disease caused by a lipid peroxidation reaction” or“lipid peroxidation reaction-induced disease” includes diseases wherethe association of the disease with the lipid peroxidation reaction isknown, for example, as shown in FIG. 1 . Examples of the diseasesinclude one or more diseases selected from the group consisting ofAlzheimer-type dementia, chronic kidney diseases, diabetic neuropathy,liver disorder, age-related macular degeneration, postischemic braindisorder, vascular dementia, arteriosclerosis, Parkinson's disease,multiple sclerosis, cancer, asthma, hypertension, cardiovasculardiseases, and age-related eye disease.

As used herein, the “subject” includes human or non-human animals.

The active drug of the present invention includes a pharmaceuticallyacceptable salt thereof. In addition, the active drug of the presentinvention or a pharmaceutically acceptable salt thereof includes ahydrate or a solvate thereof with a solvent or the like. The presentinvention also includes any form of crystal of the active drug of thepresent invention.

Examples of the pharmaceutically acceptable salt include salts withorganic bases (for example, diethanolamine salts, ethylenediaminesalts), and salts with inorganic bases (for example, salts with alkalimetals (for example, sodium, potassium) and salts with alkaline earthmetals (for example, calcium, or magnesium).

The active drug of the present invention can be administered orally orparenterally (for example, intravenously, subcutaneously, orintramuscularly, topically, rectally, transdermally, intraspinally, ornasally) as a pharmaceutical composition when used for treatment orprevention. Examples of compositions for oral administration includetablets, capsules, pills, granules, powders, solutions, and suspensions.Examples of compositions for parenteral administration include aqueousor oily injectables, ointments, creams, lotions, aerosols,suppositories, and patches. These formulations are prepared usingconventionally known techniques and can contain a non-toxic and inertcarrier or additive (hereinafter referred to as “pharmaceuticallyacceptable carrier”) which is usually used in the pharmaceutical field.

As used herein, “pharmaceutically acceptable carrier” may include, inaddition to the effective active ingredient, various active ingredientsor medicinal ingredients (including pharmacological active ingredientsand physiologically active ingredients) and additives (for example,buffering agents, isotonic agents, pH adjusters,antiseptics/preservatives, stabilizers, viscosity enhancing agents,chelating agents, surfactants, fragrances) in combination, according tovarious uses, as long as the pharmacological effect or the like is nothindered. Such ingredients can be appropriately mixed within aconcentration range that does not cause problems such as stimulation.The kinds of ingredients are not particularly limited, but examples ofthem include buffering agents (for example, sodium phosphate), isotonicagents (for example, sodium chloride), pH adjusting agents (for example,boric acid), antiseptics/preservatives (for example, benzalkoniumchloride), stabilizers (for example, mannitol), viscosity enhancingagents (for example, sodium alginate), chelating agents (for example,sodium edetate), surfactants (for example, polyoxyethylene sorbitanmonooleate), and fragrances (for example, menthol).

As used herein, the term “administering” means that an active drug orpharmaceutical composition containing it is provided and/or prescribedto an individual of subject, or the individual receives an active drugor pharmaceutical composition of the present invention. The route ofadministration of the active drug or pharmaceutical composition of thepresent invention can be any route of administration, and can varydepending on intended disease, symptom, age, weight or sex of thesubject, or the like.

As used herein, an “effective amount” means an amount of an active drugsufficient to provide the desired effect, that is, treatment orprevention of the lipid peroxidation reaction-induced diseases describedherein. The active drug or the pharmaceutical composition of the presentinvention may be used in combination with a known active drug or apharmaceutical composition for the intended disease.

The dose of the active drug of the present invention varies depending onthe individual active drug or the pharmaceutical composition, and alsodepending on the disease, age, weight, sex, or symptom of the subject,route of administration, or the like. In the case of parenteraladministration, the dose is usually 0.001 to 100 mg/kg, preferably 0.01to 100 mg/kg per day. In the case of oral administration, the dose isusually 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg per day. Theactive drug of the present invention is administered once or multipletimes (or two or three times) a day. It can also be administered onceevery several days to several weeks.

EXAMPLES

Examples of the present invention will be described below as Examples,but the present invention is not limited thereto.

Reagents, cell culture-related reagents, and profluorescent nitroxidecompounds were obtained commercially or manufactured according to knownmethods. Core Library compounds and Prestwick Chemical Library compoundswere provided from Drug Discovery Initiative, the University of Tokyo,and the Kyushu University Compound Library Drug Discovery AdvancedResearch and Education Platform Center, respectively. Commonly usedinstruments were employed as various instruments in the measurement.

Reference Example 1

Production of NBD-TEMPO Compound

2,2,6,6-Tetramethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidin-1-oxyl(NBD-TEMPO) was prepared according to the following procedure.Specifically, 366 mg (2.0 mmol) of 4-fluoro-7-nitro-2,1,3-benzoxadiazolewas dissolved in 10 mL of AcOEt, and to the resulting solution, 342 mg(2.0 mmol) of 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl was added.After stirring the mixture at room temperature for 3 hours, saturatedsaline was added, and the resulting mixture was extracted with AcOEt.The organic layer was dried over Na₂SO₄ and the solvent was completelydistilled off. Then, the residue was purified by silica gel columnchromatography (CHCl₃) to obtain 574 mg of orange yellow crystal (yield:86%). HRMS(ESI⁺) cald for C₁₅H₂₀N₅NaO₄ [M+Na]⁺: 357.1413, found:357.1415.

Reference Example 2

Production of Dansyl-TEMPO Compound

2,2,6,6-Tetramethyl-4-(5-(dimethylamino)naphthalene-1-sulfonylamino)piperidin-1-oxyl(Dansyl-TEMPO) was produced according to the method described in theliterature (for example, by Lozinsky et al.: Lozinsky, E., et. al., J.Biolchem. Biophys., Methods, 1999, 38, 29-42). Specifically, 1.03 g (6.0mmol) of 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl was dissolved in 5ml of acetone, and to the resulting solution, 1.35 g (5.0 mmol) of5-(dimethylamino)naphthalene-1-sulfonyl chloride and 0.483 ml ofpyridine were added in an ice bath. After stirring the mixture at roomtemperature overnight, saturated saline was added, and the resultingmixture was extracted with diethyl ether. The organic layer was driedover Na₂SO₄ and the solvent was completely distilled off. Then, theresidue was separated and purified by silica gel column chromatography(CHCl₃:MeOH=99:1) to obtain 396 mg of the product (yield: 20%).HRMS(ESI⁺) cald for C₂₁H₃₀N₃NaO₃S [M+Na]⁺: 427.1906, found: 427.1900.

Reference Example 3

Production of NBD-TEEPO Compound

2,2,6,6-tetraethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidin-1-oxyl(NBD-TEEPO) was produced according to the method described in theliterature (for example, by Bognar et al.: Bognar, B., et al., J.Heterocycl. Chem., 2006, 43, 81-86). Specifically, 87.6 mg (0.44 mmol)of 4-chloro-7-nitro-2,1,3-benzoxadiazole and 61 μL of Et₃N was dissolvedin 10 mL of AcOEt, and to the resulting solution, 100 mg (0.44 mmol) of4-amino-2,2,6,6-tetraethylpiperidin-1-oxyl was added. After stirring themixture at room temperature for 6 hours, saturated saline was added, andthe resulting mixture was extracted with AcOEt. The organic layer wasdried over Na₂SO₄ and the solvent was completely distilled off. Then,the residue was separated and purified by silica gel columnchromatography (Hexane:AcOEt=100:0 to 70:30) to obtain 83 mg of orangeyellow crystal (yield: 6%). HRMS(ESI⁺) cald for C₁₉H₂₈N₅NaO₄ [M+Na]⁺:413.2034, found: 413.2024.

Reference Example 4

Production of NBD-Pen Compound

Similar to the synthesis method of2,2,6,6-tetraethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)piperidin-1-oxyl(NBD-TEEPO), but replacing 100 mg (0.44 mmol) of4-amino-2,2,6,6-tetraethylpiperidin-1-oxyl (4) with 100 mg (0.44 mmol)of 4-amino-2,2,6-trimethyl-6-pentylpiperidin-1-oxyl (8), the reactionwas carried out. The product was separated and purified by silica gelcolumn chromatography (Hexane:AcOEt=100:0 to 70:30) to obtain 83 mg oforange yellow crystal (yield: 48%). HRMS(ESI⁺) cald for C₁₉H₂₈N₅NaO₄[M+Na]⁺: 413.2034, found: 413.2056.

Example 1

Evaluation of Responsiveness of Profluorescent Nitroxide Probe to LipidPeroxidation Reaction

In a phosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5% acetonitrile),profluorescent nitroxide (NBD-TEMPO compound or Dansyl-TEMPO compound)(5.0 μM) and liposomes (2.5 mg/mL Egg PC, 0.1 mg/mL DCP) were mixed at37° C. AAPH (20 mM) was added to the mixture, and the lipid peroxidationreaction was started. After 40 minutes, the fluorescence intensity wasmeasured at an excitation wavelength of 470 nm and a fluorescencewavelength of 530 nm for NBD-TEMPO, and at an excitation wavelength of300 nm and a fluorescence wavelength of 500 nm for Dansyl-TEMPO.

The results are shown in FIG. 2 .

When the prepared liposomes were stimulated with addition of AAPH, thefluorescence intensity of NBD-TEMPO increased 8.2 times compared to thatin the case without AAPH, while the fluorescence intensity ofDansyl-TEMPO increased only 1.4-fold. Thus, the NBD group was employedas the fluorophore.

Example 2

Evaluation of Reactivity of Profluorescent Nitroxide Probe with VariousReductants

Profluorescent nitroxides (5.0 μM) (NBD-TEMPO compound, NBD-Pencompound, or NBD-TEEPO compound) and 50 μM of various reductants (AsA,UA, TPL, Eda, Catechin, Trolox) were mixed in phosphate buffer (10 mM,pH 7.4, 0.5% DMSO, 0.5% acetonitrile) containing liposomes (2.5 mg/mLEgg PC, 0.1 mg/mL DCP) at 37° C. Lipid peroxidation reaction was causedby adding AA (0.5 mM) and LOX (25 μg/mL). After 40 minutes, thefluorescence intensity was measured at an excitation wavelength of 470nm and a fluorescence wavelength of 530 nm. Here, AsA means ascorbicacid, UA means uric acid, TPL means 2,2,6,6-tetramethylpiperidin-1-oxyl,Eda means Edaravone, catechin means (−)-epicatechin, and Trolox means6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid.

The results are shown in FIGS. 3A-FIG. 3B.

As model antioxidants, the following six compounds were selected (FIG.3A). AsA, which acts as a water-soluble antioxidant in human body and isalso included in many foods or the like; Uric acid, the most abundantantioxidant in human blood;4-hydroxy-2,2,6,6-tetramethylpiperidyl-1-oxyl (Tempol), a compound whichis widely used in research using laboratory animal models amongnitroxide compounds due to its low toxicity; Edaravone (Eda), which isapproved as a free radical scavenger; Catechin ((−)-epicatechin), whichis included in tea, wine or the like, and commonly consumed; and Trolox,an analog compound of VitE. It is known that the water-octanol partitioncoefficients (Log Po/w) of these six compounds are high in the order ofAsA<UA<TPL<Eda<Catechin<Trolox.

When the NBD-TEMPO compound was used, the fluorescence intensityincreased about 7.7 times by the reaction with AsA, and when the NBD-Pencompound was used, the fluorescence intensity increased about 2.9 timesby the reaction with Eda. However, when the NBD-TEEPO compound was used,the fluorescence intensity had little increase (FIG. 3B).

Example 3

Evaluation of Reactivity of Profluorescent Nitroxide Probe with VariousOxidants

Profluorescent nitroxides (NBD-TEMPO compound, NBD-Pen compound, orNBD-TEEPO compound) (5.0 μM) and various oxidants were mixed inphosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5% acetonitrile) at 37° C.Hydrogen peroxide, hypochlorous acid, and potassium oxide (0.5 mM each)were used as the oxidants. •OH was generated with hydrogen peroxide (0.5mM) and FeSO₄ (5.0 μM). Lipid radicals were generated by liposomes (2.5mg/mL Egg PC, 0.1 mg/mL DCP) and AAPH (10 mM). After 30 minutes,fluorescence intensity was measured at an excitation wavelength of 470nm and a fluorescence wavelength of 530 nm.

The results are shown in FIG. 4 .

Any probe molecules showed little increase of the fluorescence intensityby reaction with ROS. The NBD-TEMPO compound had the lowestresponsiveness to lipid peroxidation reaction, thus it was found thatthe NBD-TEEPO and NBD-Pen compounds have advantages in responsiveness tolipid peroxidation reaction.

From the above results, it was decided that the NBD-TEEPO compound whichshowed the highest reduction resistance and high responsiveness to lipidperoxidation reaction be employed.

Example 4

Evaluation of Reaction Initiator Concentration-Dependent LipidPeroxidation in Artificial Lipid Membranes (NBD-TEEPO Assay)

Probes (NBD-TEEPO compound) (5.0 μM) and liposomes (2.5 mg/mL Egg PC,0.1 mg/mL DCP) were mixed in phosphate buffer (10 mM, pH 7.4, 0.5% DMSO,0.5% acetonitrile) at 37° C. AAPH (0-20 mM) or FeSO₄ (0-2.0 mM) wasadded to the mixture, and the lipid peroxidation reaction was started.After 40 minutes in the AAPH system or after 60 minutes in the Fe²⁺system, the fluorescence intensity was measured at an excitationwavelength of 470 nm and a fluorescence wavelength of 530 nm.

The results are shown in FIG. 5A-FIG. 5D 5 (AAPH) and FIGS. 6A-FIG. 6D(FeSO₄).

The fluorescence intensity of the probe increasedconcentration-dependently in both of the AAPH and Fe²⁺ systems (FIG. 5Aand FIG. 6A). Addition of water-soluble AsA in the AAPH system orlipid-soluble antioxidant Eda in the Fe²⁺ system inhibited thefluorescence increase concentration-dependently (FIG. 5B and FIG. 6B).Thus, the lipid peroxidation inhibitory effects of six typicalantioxidants were measured. When compared the measurement result withthe result of the TBARS method, an existing method shown in Example 5(FIG. 5D and FIG. 6D), both showed the same tendency (FIG. 5C and FIG.6C).

For the cell-free based assay system using AAPH or Fe²⁺, the S/B ratios,CV values, and Z′-factors, which represent the quality of assay system,were examined using the NBD-TEEPO compound. The results are shown in thetable below. All indicators exceeded the target values (Table 1).

TABLE 1 AAPH Fe²⁺ Indicators system system Target value S/B ratio 18.78.7 2 or more CV value of Background (%) 4.9 3.2 10% or less CV value ofControl(%) 5.4 3.2 10% or less Z′-factor 0.82 0.88 0.5 or more

Example 5

Evaluation of Lipid Peroxidation Inhibition by Antioxidants inArtificial Lipid Membranes (TBARS Assay)

Various reductants (10 μM) and liposomes (2.5 mg/mL Egg PC, 0.1 mg/mLDCP) were mixed in phosphate buffer (10 mM, pH 7.4, 0.5% DMSO, 0.5%acetonitrile) at 37° C. AAPH (20 mM) or FeSO₄ (1.0 mM) was added to themixture, and the lipid peroxidation reaction was started. After 60minutes, the lipid peroxidation reaction was stopped by BHT (10 mM).Acetic acid (5.7%), TBA (0.56%), and SDS (1.07%) were added, and afterstirring, the mixture was allowed to react at 60° C. for 60 minutes. Theresultant was subjected to centrifugation (2000 rpm, 4° C., 15 minutes),and the fluorescence intensity was measured at an excitation wavelengthof 512 nm and a fluorescence wavelength of 553 nm.

In the same manner as in Example 4, lipid peroxidation inhibitioneffects of the six typical antioxidants were measured.

The results are shown in FIGS. 5A-FIG. 5D (AAPH) and FIG. 6A-FIG. 6D(FeSO₄). The lipid peroxidation inhibitory effects shown in the Figureshad a similar tendency to the result of Example 4.

Example 6

Cell Culturing

Human hepatoma cells (HepG2 cells) were cultured with DMEM medium(containing 10% FBS, 1% Penicillin-Streptomycin and 1×MEM non-essentialamino acids) in a CO₂ incubator (37° C., 5% CO₂). Passaging wasperformed when 60-70% subconfluent state was reached. DMEM media (phenolred free, containing 1% Penicillin-Streptomycin) was used for variousmeasurements.

Example 7

Evaluation of Reaction Initiator Concentration-Dependent IntracellularLipid Peroxidation Reaction (NBD-TEEPO Assay)

HepG2 cells were seeded in a 96-well plate at 10,000 cells/well. Thecells were incubated for 24 hours to adhere. AA (0 to 200 μM) or tBHP (0to 300 μM), and probe (5.0 μM) were added to the cells in DMEM medium(0.5% DMSO, 0.5% acetonitrile), and after 45 minutes, the fluorescenceintensity was measured at an excitation wavelength of 470 nm and afluorescence wavelength of 530 nm.

Example 8

Evaluation of Intracellular Lipid Peroxidation Inhibition byAntioxidants (NBD-TEEPO Assay)

HepG2 cells were seeded in a 96-well plate at 10,000 cells/well. Thecells were incubated for 24 hours to adhere. AA (200 μM) or tBHP (0 to300 μM), antioxidant (50 μM), and probe (5.0 μM) were added to the cellsin DMEM medium (0.5% DMSO, 0.5% acetonitrile), and after 45 minutes, thefluorescence intensity was measured at an excitation wavelength of 470nm and a fluorescence wavelength of 530 nm.

The results are shown in FIGS. 7A-FIG. 7E.

AA and tBHP concentration-dependent fluorescence increases were observed(FIG. 7A and FIG. 7C). Furthermore, addition of antioxidants inhibitedthe increase (FIG. 7B and FIG. 7D). Catechin showed the strongestinhibitory effect, and the inhibitory effect had the same tendencyregardless of the reaction initiator.

Example 9

Evaluation of Changes in Reaction Initiator Concentration-Dependent CellViability (MTT Assay)

HepG2 cells were seeded in a 96-well plate at 10,000 cells/well. Thecells were incubated for 24 hours to adhere. AA (0 to 100 μM) or tBHP (0to 100 μM) were added to the cells in DMEM medium (0.5% DMSO, 0.5%acetonitrile), and after 24 hours, the medium was exchanged. MTTsolution (0.5 mg/mL, 0.5% DMSO) was added, and then the cells wereincubated for 4 hours, and the solution was removed. 100 μL of DMSO wasadded, and the absorbance at 630 nm was measured. The cell viability wascalculated with regarding the case without AA or tBHP as 100%.

Example 10

Evaluation of Changes in Cell Viability by Antioxidants (MTT Assay)

HepG2 cells were seeded in a 96-well plate at 10,000 cells/well. Thecells were incubated for 24 hours to adhere. AA (200 μM) or tBHP (0 to300 μM), and antioxidant (50 μM) were added to the cells in DMEM medium(0.5% DMSO, 0.5% acetonitrile), and the medium was changed after 24hours. MTT solution (0.5 mg/mL, 0.5% DMSO) was added, and the cells wereincubated for 4 hours, then the solution was removed. 100 μL of DMSO wasadded, and the absorbance at 630 nm was measured. The cell viability wascalculated with regarding the case without AA or tBHP as 100%.

The results are shown in FIGS. 8A-FIG. 8E.

By the addition of AA and tBHP, the cell viability decreasedconcentration-dependently (FIG. 8A and FIG. 8C). Furthermore, the effectwas inhibited by antioxidants (FIG. 8B and FIG. 8D). Depending on thereaction initiators, there were different tendencies in the antioxidantshaving high inhibitory effect.

For the cell-based assay system using AA or tBHP, the S/B ratios, CVvalues, and Z′-factors, which represent the quality of assay system,were examined using the NBD-TEEPO compound or MTT. The results are shownin Tables 2 and 3 below. All indicators exceeded the target values.

TABLE 2 NBD-TEEPO compound AA tBHP Indicators system system Target valueS/B ratio 4.1 2.2 2 or more CV value of Background (%) 6.5 3.8 10% orless CV value of Control (%) 2.8 5.0 10% or less Z′-factor 0.83 0.62 0.5or more

TABLE 3 MTT method AA tBHP Indicators system system Target value S/Bratio 25.0 6.3 2 or more CV value of Background (%) 4.7 6.9 10% or lessCV value of Control (%) 4.8 6.9 10% or less Z′-factor 0.85 0.71 0.5 ormore

Example 11

Primary Screening Using Core Library of Drug Discovery Initiative, theUniversity of Tokyo (AAPH System)

For the compounds, 2 mM 100% DMSO solutions (dispensed at 0.125 μL/well)were provided from Drug Discovery Initiative, the University of Tokyo.Solution A containing liposomes (5.0 mg/mL Egg PC, 0.2 mg/mL DCP) andprobe (10 μM) in phosphate buffer (10 mM, pH 7.4, 1.0% acetonitrile) andSolution B containing AAPH (40 mM) in phosphate buffer (10 mM, pH 7.4)were prepared. 12.5 μL each of solutions A and B were dispensed withMultidrop Combi. The final concentration was liposomes (2.5 mg/mL EggPC, 0.1 mg/mL DCP), 5.0 μM NBD-TEEPO compound, 50 μM test compound and20 mM AAPH in phosphate buffer (10 mM, pH 7.4, 0.5% acetonitrile, 0.5%DMSO). The reaction mixture was mixed at 37° C., and after 40 minutes,the fluorescence intensity at an excitation wavelength of 470 nm and afluorescence wavelength of 530 nm was measured. The activity values ofeach test compound were determined according to the expression describedherein.

The results are shown in FIG. 10 .

Of 9600 compounds, 1858 compounds had activity values below 0, that is,did not inhibit the lipid peroxidation reaction. On the other hand, 7711compounds inhibited the lipid peroxidation reaction, and 836 compoundsof which exhibited higher activity values than the known compoundEdaravone. These 836 compounds were decided as hit compounds (candidatecompounds) in the primary screening, and proceeded to the evaluation inFe²⁺ system.

Example 12

Primary Screening Using Core Library of Drug Discovery Initiative, theUniversity of Tokyo (FeSO₄ System)

Subsequently, evaluation was performed in Fe²⁺ system for the 836compounds that exhibited higher lipid peroxidation inhibitory effectsthan Edaravone in the AAPH system. For the compounds, 2 mM 100% DMSOsolutions (dispensed at 0.2 μL/well) were provided from Drug DiscoveryInitiative, the University of Tokyo. Solution A containing liposomes(2.78 mg/mL Egg PC, 0.11 mg/mL DCP) and 5.6 μM probe in phosphate buffer(10 mM, pH 7.4, 0.56% acetonitrile) and Solution B containing 10 mMFeSO₄ in distilled water were prepared. Solution A was dispensed by 36μL with Multidrop Combi. Solution B was dispensed by 4 μL with BiomekNXP. The final concentration was liposomes (2.5 mg/mL Egg PC, 0.1 mg/mLDCP), 5.0 μM probe, 50 μM test compound, 1.0 mM FeSO₄ in phosphatebuffer (10 mM, pH 7.4, 0.5% acetonitrile, 0.5% DMSO). The reactionmixture was mixed at 37° C., and the fluorescence intensity at anexcitation wavelength of 470 nm and a fluorescence wavelength of 530 nmwas measured over time every 3 minutes. AUC was calculated from thefluorescence intensity for 180 minutes, and the activity value of eachtest compound was calculated according to the expression describedherein (FIG. 11 ).

The results are shown in FIG. 12 .

Of the 836 compounds, 268 compounds had activity values below 0, thatis, did not inhibit the lipid peroxidation reaction. On the other hand,568 compounds inhibited lipid peroxidation reaction. Of these, 197compounds showed higher activity than Edaravone. The top 80 compoundswith higher inhibitory effect were decided as hit compounds (candidatecompounds), and proceeded to secondary screening.

Example 13

Secondary Screening Using Core Library of Drug Discovery Initiative, theUniversity of Tokyo (NBD-TEEPO Assay)

Secondary screening was performed for 80 compounds selected in theprimary screening.

For the compounds, 10 mM 100% DMSO solutions (dispensed at 5.0 μL/well)were provided from Drug Discovery Initiative, the University of Tokyo.495 μL of DMEM medium for measurement was added, and a 1.0% DMSOsolution containing 100 μM of the compound was prepared. This solutionwas dispensed by 80 μL with Biomek NXP into a measuring plate on whichcells were seeded in advance. To the test compound (100 μM 1.0% DMSO),64 μL of DMEM medium (1.25% acetonitrile) containing 12.5 μM NBD-TEEPOcompound, and 16 μL of PBS in which AA (2000 μM, 5.0% ethanol) or tBHP(3000 μM) was dissolved were manually dispensed. The final concentrationwas 50 μM test compound and 200 μM AA or 300 μM tBHP in DMEM medium(0.5% DMSO, 0.5% acetonitrile).

The reaction mixture was mixed at 37° C., and the fluorescence intensityat an excitation wavelength of 470 nm and a fluorescence wavelength of530 nm was measured over time every 3 minutes. The AUC was calculatedfrom the fluorescence intensity for 45 minutes in the AA-added system or60 minutes in the tBHP-added system, and the activity value of each testcompound was calculated according to the expression described herein.

The results are shown in FIGS. 13A-FIG. 13D.

In the AA-added system, 40 compounds of the 80 compounds had activityvalues below 0%, that is, did not inhibit the lipid peroxidationreaction in the cultured cell system. On the other hand, 40 compoundsinhibited lipid peroxidation reaction. Of these, 32 compounds showedhigher activity than Edaravone (FIG. 13A). In the tBHP-added system, 17compounds of the 80 compounds were below the activity value of 0%, while63 compounds inhibited the lipid peroxidation reaction. Of these, 31compounds showed higher activity than Edaravone (FIG. 13B). The activityvalues in the two systems were plotted (FIG. 13C), and the fourcompounds that showed high activity values in both systems, CompoundNos. 7, 48, 64 and 80, were decided as hit compounds (FIG. 13D), andproceed to tertiary screening.

Compound 7:

-   2-((4-(Phenylamino)phenyl)amino)-N-(4-sulfamoylphenyl)propanamide    Compound 48:-   2,6-Dimethoxy-4-(2-(8-nitroquinolin-2-yl)vinyl)phenol    Compound 64:-   N²,N²-Dimethyl-9H-fluorene-2,3-diamine    Compound 80:-   N-(3-Methoxy-4-((3-methyl-1-10H-indolo[3,2-b]quinolin-11-yl)amino)phenyl)methanesulfonamide

Example 14

Secondary Screening Using Core Library of Drug Discovery Initiative, theUniversity of Tokyo (MTT Assay)

Secondary screening was performed for 80 compounds selected in theprimary screening.

For the compounds, 10 mM 100% DMSO solutions (dispensed at 5.0 μL/well)were provided from Drug Discovery Initiative, the University of Tokyo.495 μL of DMEM medium for measurement was added, and 1.0% DMSO solutioncontaining 100 μM of the compound was prepared. This solution wasdispensed by 80 μL with Biomek NXP into a measuring plate on which cellswere seeded in advance. To the test compound (100 μM 1.0% DMSO), 64 μLof DMEM medium, 16 μL of PBS in which AA (1000 μM, 5.0% ethanol) or tBHP(1000 μM) was dissolved were manually dispensed. The final concentrationwas 5.0 μM NBD-TEEPO compound, 50 μM test compound, and 100 μM AA ortBHP in DMEM medium (0.5% DMSO, 0.5% acetonitrile). After 24 hours, themedium was exchanged, and MTT solution (0.5 mg/mL, 0.5% DMSO) was added.Then, cells were incubated for 24 hours, and the solution was removed.100 μL of DMSO was added, and the absorbance at 630 nm was measured. Thecell viability was calculated according to the expression describedherein, with regarding the case without AA or tBHP as 100%.

The results are shown in FIGS. 14A-FIG. 14D.

In the AA-added system, 6 compounds of the 80 compounds had activityvalues below 0%, that is, did not inhibit cell death caused by AAstimulation. On the other hand, 74 compounds inhibited cell death causedby AA stimulation. Of these, 64 compounds showed higher activity thanEdaravone (FIG. 14A). In the tBHP-added system, 8 compounds of the 80compounds were below the activity value of 0%, while 73 compoundsinhibited cell death caused by tBHP stimulation. Of these, 14 compoundsshowed higher activity than Edaravone (FIG. 14B). The inhibition ratesin the two systems were plotted (FIG. 14C), and five compounds whichshowed high inhibition rates in both systems, Compound Nos. 19, 39, 52,73, and 78, were decided as hit compounds (FIG. 14D), and proceeded totertiary screening.

Compound 19:

-   N-(2-Chlorophenyl)-5-(2-(1-pyridin-2-yl)ethylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine    Compound 39:-   1-(7,7-Dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl)-N-(1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)-N-methylmethanesulfonamide    Compound 52:-   Methyl 3-amino-4-(phenylamino)benzoate    Compound 73:-   3-(2-(3-(2-Hydroxyethoxy)phenyl)-3-((2-morpholinoethyl)amino)imidazo[1,2-a]pyridin-6-yl)benzamide    Compound 78:-   1-(4-(Trifluoromethoxy)phenyl)indolin-5-amine

Example 15

Tertiary Screening Using Core Library of Drug Discovery Initiative, theUniversity of Tokyo (NBD-TEEPO Assay)

The Core Library of Drug Discovery Initiative, the University of Tokyo,includes structural analogs for each compound. Then, secondary screeningwas also performed for structural analog compounds for each candidatecompound in the same manner as Example 13 and the activity values wereevaluated.

Specifically, the nine hit compounds, Compound Nos. 7, 19, 39, 48, 52,64, 73, 78, 80, of the secondary screening had 5, 2, 4, 6, 6, 5, 5, 10,2 structural analogs, respectively, thus, a total 45 structural analogswere taken. Then, including the original 9 compounds, a total 54compounds were subjected to tertiary screening, and the activity valueswere calculated in the same manner.

The results are shown in FIGS. 15A-FIG. 15D, FIGS. 16A-FIG. 16D and FIG.17 .

1) First, in the assay using NBD-TEEPO, when the AA-added system wasused, 15 compounds out of 54 compounds had activity values below 0%,that is, did not inhibit lipid peroxidation reaction, while 39 compoundsinhibited lipid peroxidation reaction. Of these, 33 compounds showedhigher activity than Edaravone (FIG. 15A). When the tBHP-added systemwas used, 9 compounds of the 54 compounds were below the activity valueof 0%, while 45 compounds inhibited lipid peroxidation reaction. Ofthese, 41 compounds showed higher activity than Edaravone (FIG. 15B).2) Then, in the MTT assay, when the AA-added system was used, 7 out of54 compounds had activity values below 0%, that is, did not inhibit celldeath caused by AA stimulation, while 47 compounds inhibited cell deathcaused by AA stimulation. Of these, 31 compounds showed higher activitythan Edaravone (FIG. 16A). When the tBHP-added system was used, 17compounds of the 54 compounds were below the activity value of 0%, while37 compounds inhibited cell death caused by tBHP stimulation. Of these,37 compounds showed higher activity than Edaravone (FIG. 18B).3) Then, test compounds alone were further incubated for 72 hours, andevaluation of cytotoxicity thereof was performed. For the evaluation ofcytotoxicity, 50 μM antioxidant was added to HepG2 cells in DMEM medium(0.5% DMSO), and after incubation at 37° C. for 72 hours, the cellviability was measured.4) The activity values of the two-added systems in each of the assayusing NBD-TEEPO and the MTT assay were plotted (FIG. 15C and FIG. 16C).As the result, Compound No. 52 analogs (Compound Nos. 52, 52-1, 52-3,52-4, 52-5) and Compound No. 78 analogs (Compound Nos. 78, 78-3, 78-4,78-5, 78-6, 78-8) showed high lipid peroxidation inhibitory effects andcell death inhibitory effects (FIG. 15D and FIG. 16D).

Compound No. 80 analogs (Compound Nos. 80 and 80-2) showed the highestlipid peroxidation inhibitory effect, but cytotoxicity of the compoundswas extremely high (FIG. 17 ).

Among the compound No. 52 and its analogs (Compound Nos. 52-1 to 52-6),those that exhibited lipid peroxidation inhibitory effects have askeleton A represented by the structure below as a common structure. Theskeleton A has been reported to have antioxidant activity (Hu ML., etal., Nutr. Biochem., 1995, 6, 504-508).

Among compounds No. 78 and its analogs (Compound Nos. 78-1 to 78-10),those that exhibited lipid peroxidation inhibitory effects have askeleton B represented by the structure below as a common structure.

From the above results, it is suggested that compounds having theskeleton A or B, which is a common structure of compounds found ascandidate compounds by the screening of the present invention, are verylikely to be prominent as lipid peroxidation inhibitors.

Compound 52-1:

-   Methyl 3-amino-4-((4-methoxyphenyl)amino)benzoate    Compound 52-2:-   Methyl 3-amino-4-((2-methoxyphenyl)amino)benzoate    Compound 52-3:-   Methyl 3-amino-4-((3-methoxyphenyl)amino)benzoate    Compound 52-4:-   Methyl 3-amino-4-(benzylamino)benzoate    Compound 52-5:-   Methyl 3-amino-4-((1-phenylethyl)amino)benzoate    Compound 52-6:-   N-(2-(phenylamino)phenyl)acetamide

Compound 78-1:

-   1-(3,5-dimethylphenyl)indolin-2,3-dione    Compound 78-2:-   1-(3,5-Dimethylphenyl)-3,3-difluoroindolin-2-one    Compound 78-3:-   1-(3,5-Dimethylphenyl)-1H-indol-6-amine    Compound 78-4:-   1-(3,5-Dimethylphenyl)indolin-6-amine    Compound 78-5:-   1-(4-Methoxyphenyl)-1H-indol-5-amine    Compound 78-6:-   1-(4-(Methylthio)phenyl)-1H-indol-5-amine    Compound 78-7:-   1-(4-(Trifluoromethyl)phenyl)-1H-indol-5-amine    Compound 78-8:-   1-(4-(Trifluoromethoxyphenyl)-1H-indol-5-amine    Compound 78-9:-   1-(4-(Methylthio)phenyl)indolin-5-amine    Compound 78-10:-   1-(4-(Trifluoromethyl)phenyl)indolin-5-amine

Example 16

Prestwick Chemical Library Primary Screening (AAPH System)

In the primary screening, in the AAPH and Fe²⁺ systems, measurement wasconducted for 1280 compounds as test compounds, respectively. Theexperiment method and the calculation method of the activity value wereperformed in the same manner as in the above case in which the CoreLibrary of Drug Discovery Initiative, the University of Tokyo, was used.For the test compounds, solutions diluted to 20 μM (dispensed at 20μL/well) in phosphate buffer (10 mM, pH 7.4, 2% DMSO) were provided fromthe Kyushu University Compound Library Drug Discovery Advanced Researchand Education Platform Center.

First, when the AAPH system assay was used, Solution A containingliposomes (10 mg/mL Egg PC, 0.4 mg/mL DCP) and NBD-TEEPO compound (20μM) in phosphate buffer (10 mM, pH 7.4, 2.0% acetonitrile) and SolutionB containing AAPH 80 mM in phosphate buffer (10 mM, pH 7.4) wereprepared. 10 μL each of solutions A and B were dispensed with MultidropCombi. The final concentration was liposomes (2.5 mg/mL Egg PC, 0.1mg/mL DCP), 5.0 μM NBD-TEEPO compound, 10 μM test compound, and 20 mMAAPH in phosphate buffer (10 mM, pH 7.4, 0.5% acetonitrile, 1% DMSO).After 40 minutes at 37° C., the fluorescence intensity at an excitationwavelength of 470 nm and a fluorescence wavelength of 530 nm wasmeasured.

Example 17

Prestwick Chemical Library Primary Screening (FeSO₄ System)

Then, Fe²⁺ system assay was used, and solution A containing liposomes(10 mg/mL Egg PC, 0.4 mg/mL DCP) and 20 μM of NBD-TEEPO compound inphosphate buffer (10 mM, pH 7.4, 2.0% acetonitrile) and Solution Bcontaining 4.0 mM FeSO₄ in distilled water were prepared. 10 μL each ofsolutions A and B were dispensed with Multidrop Combi. The finalconcentration was liposomes (2.5 mg/mL Egg PC, 0.1 mg/mL DCP), 5.0 μMNBD-TEEPO compound, 10 μM test compound and 1.0 mM FeSO₄ in phosphatebuffer (10 mM, pH 7.4, 0.5% acetonitrile, 1% DMSO). After 180 minutes at37° C., the fluorescence intensity at an excitation wavelength of 470 nmand a fluorescence wavelength of 530 nm was measured.

The results are shown in FIGS. 18A-FIG. 18B and FIGS. 19A-FIG. 19C.

1) As a result, in the AAPH system, 330 compounds of the 1280 compoundshad activity values below 0, that is, did not inhibit lipid peroxidationreaction, while 950 compounds inhibited lipid peroxidation reaction. Ofthese, 190 compounds showed higher activity values than the knowncompound Edaravone (FIG. 18A). In the Fe²⁺ system, 434 compounds of the1280 compounds had activity values below 0, while 846 compoundsinhibited lipid peroxidation reaction. Of these, 19 compounds showedhigher activity values than the known compound Edaravone (FIG. 18B).

From the combined results in the AAPH and Fe²⁺ systems, 16 hit compoundswere obtained (FIG. 19A and FIG. 19C).

2) Since the Prestwick Chemical Library is a compound library ofcompounds with known pharmacological activity, information on the actionpoint, pharmacokinetics, safety and the like can be obtained from thedatabase and bibliographic information provided by this library. Basedon these information, narrowing of the 16 hit compounds was performed.These hit compounds included therapeutic drugs of a wide range ofdisease areas such as the cardiovascular system, the central nervoussystem, the respiratory system, and antibacterial drugs (FIG. 19B).

(Pharmacological Test)

Pharmacological activity for age-related macular degeneration (AMD) wasexamined.

A test was performed using a light irradiation model, which is widelyemployed as an atrophic AMD model mouse, as a test model. Test compoundswere narrowed down from the 16 candidate compounds selected by thescreening for the Prestwick Chemical Library to the following 5compounds (Compound V, Compound W, Compound X, Compound Y, Compound Z)which have been reported to be highly permeable to blood-retinal barrier(BRB), and the pharmacological activity thereof was examined.

Compound V:

-   Apomorphine ((R)-(−)-apomorphine hydrochloride)

This compound is known as an anti-Parkinson drug that acts on dopamineD₁D₂ receptors.

Compound W:

-   Etheroline ((−)-eseroline fumarate)

This compound is known to act on opioid receptors to have analgesiceffects.

Compound X:

-   Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline)

This compound is known to have an antioxidant action.

Compound Y:

-   Methyldopa (Methyldopa sesquihydrate)

This compound is known as a blood pressure lowering drug that acts onadrenergic α₂ receptors.

Compound Z:

-   Olanzapine    (2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine

This compound is known as an antipsychotic drug that acts on manyreceptors.

(Procedures)

First, AMD model mice were produced according to the following schedule.

Laboratory Animals

Male BALB/c mice (4 weeks old) were purchased from Japan SLC, Inc., andallowed to acclimate for a week before being used for experiments.Laboratory animal chow (CLEA Rodent Diet CE-2, CREA JAPAN, INC.) wasused as food, and tap water was freely consumed as drinking water. Theanimals were raised under light and dark cycles of every 12 hours. Allanimal experiments were conducted under the approval of the KyushuUniversity Animal Experiment Committee.

Production of Light-Induced AMD Model Mice

To BALB/c mice, 5 mL/kg of 10 mM compound dissolved in PBS containing10% polyethylene glycol (PEG) 300 was intraperitoneally administered.Thirty (30) minutes later, a drop of Midrin P (5 mg/mL tropicamide, 5mg/mL phenylephrine hydrochloride; Santen Pharmaceutical Co., Ltd.) wasapplied to each eye as a mydriatic. The mice were irradiated with 8000lux white light for 10 hours, then returned to under normal light anddark cycles, and raised for 6 days. On day 7, the animal was euthanizedby cervical dislocation, and the eyeballs were removed (FIG. 20A).

Preparation of Frozen Eyeball Section

10 mM of the test compound was dissolved in PBS containing 10% PEG 300,and the obtained solution was administered once to male BALB/c miceintraperitoneally at a dose of 5 mL/kg. From thirty minutes after theadministration, the mice were irradiated with 8000 lux white light for10 hours. Then, they were raised under normal light and dark cycles for6 days. On day 7, the animals were euthanized, then the eyeballs wereremoved. Frozen sections of 8 μm thickness were prepared and subjectedto hematoxylin eosin (HE) staining, and the thickness of the outernuclear layer (ONL) was measured over 27 points every 180 μm (FIG. 20B).

HE Staining

The preparation was air-dried for 1 hour, fixed with acetone at roomtemperature for 15 minutes, then immersed in 99.5% EtOH, 80% EtOH, 70%EtOH, and purified water in this order for 3 minutes each, and stainedwith hematoxylin for 10 minutes. Then, it was washed with running waterfor 10 minutes, soaked in warm water for 1 minute, and stained witheosin for 1.5 minutes. After washing with purified water, it wasimmersed in 70% EtOH, 80% EtOH, and 99.5% EtOH in this order for 3minutes each, then washed with xylene three times, dried, and thenenclosed with VectaMount™ Mounting Medium. The resultant was subjectedto observation and imaging with Keyence fluorescence microscope(BZ-9000).

Statistical Analysis

Results were expressed as mean+standard deviation. Dunnett's Test wasused for multigroup comparison.

(Results)

The imaging results are shown in FIGS. 21A-FIG. 21C. The figures includean inner nuclear layer (INL) in the top, an outer nuclear layer (ONL) inthe middle, and a retinal pigment epithelium (RPE) in the bottom. Whencell death occurs due to lipid peroxidation, the thickness of the outernuclear layer in the middle becomes thinner.

First, the thickness of ONL was significantly reduced by lightirradiation (see, FIG. 21A negative control). The extent of ONLdisorders was particularly severe on the superior side of the eyeball(FIG. 21B), and these results were consistent with the results knownfrom the literature (see, for example, Tanito M., et al., Invest.Ophthalmol. Vis. SCI., 2007, 48 (4), 1900-5.).

On the other hand, in the case of the five test compounds used in thisstudy, the ONL thickness in either case did not differ so much comparedto that of the positive control, and significant thicknesses wereobserved compared to that of Edaravone and OT-551 as control compoundseven at the same dose of 50 μmol/kg (FIG. 21C).

For OT-551, which is a compound known to have a high retinal protectiveeffect, approximately 100 mg/kg (360 μmol/kg) has been reported to berequired in a light irradiation model mouse. Thus, the dose 50 μmol/kgin this study, about one-seventh of that of OT-551, is a considerablylow dose.

In addition, 50 μmol/kg is less than one-tenth of each median lethaldose (LD50) of the five test compounds, thus the compounds have beenconfirmed to be safe (see FIG. 22 ).

From the above results, it was suggested that the compounds selected bythe screening of the present invention are useful compounds forage-related macular degeneration.

INDUSTRIAL APPLICABILITY

According to the assay method, the assay kit, and the screening reactionusing the fluorophore compound of the present invention, it is easy toexplore a compound having lipid peroxidation inhibitory activity.Furthermore, candidate compounds according to the screening methods ofthe present invention are useful for treating lipid peroxidationreaction-induced diseases, such as age-related eye diseases.

The invention claimed is:
 1. An assay kit for detecting lipidperoxidation inhibitory activity of a test compound, comprising: acompound represented by formula (I):

a liposome, and at least one compound selected from the group consistingof 2,2′-azobis(2-aminopropane) dihydrochloride and a divalent iron ionsource material in a buffer.
 2. The assay kit according to claim 1,wherein the divalent iron ion source material is iron(II) sulfate. 3.The assay kit according to claim 1, comprising a package insert showingan activity value of a compound having lipid peroxidation inhibitoryactivity.
 4. An assay kit comprising any two or more of the assay kitsaccording to claim 1.